Extract of e. coli cells having mutation in ribosomal protein s12, and method for producing protein in cell-free system using the extract

ABSTRACT

It is intended to provide an extract, kit and a process for synthesizing a protein in a cell-free system. The extract is characterized by being prepared from mutant  Escherichia coli  cells having a mutation in the ribosomal protein S12 gene. In a preferred embodiment, the mutation is such a mutation as conferring a resistance or dependence to streptomycin on  E. coli  whereby the reading efficiency of mRNA codon on ribosomes can be improved and thus the protein productivity can be elevated.

This application is a Continuation of co-pending application Ser. No.11/137,395 filed on May 26, 2005, and for which priority is claimedunder 35 U.S.C. §120; and this application claims priority ofApplication No. PCT/JP2003/015136 filed on Nov. 27, 2003, which alsoclaims priority to JP Patent Application No. 2002-345597 filed on Nov.28, 2002. The entire contents of these applications are incorporated byreference.

TECHNICAL FIELD

The present invention relates to an extract prepared from mutant E. colicells having a mutation in the ribosomal protein S12 gene, and to amethod for producing a protein in a cell-free system using the extract.

BACKGROUND ART

A cell-free protein synthesis system using a cell extract is utilizedmainly for identification of gene products and for investigation oftheir activities. For example, the system enables the analysis offunctions of synthesized proteins such as the enzymatic activity and DNAbinding capability, or the determination of the molecular weight oftranslated products by labeling them with radioisotopes. Recently,techniques of drastically increasing the production amount in the systemhave been developed, and the system has become utilized also for proteinstructure analysis through X-ray crystallography, NMR and the like.

For extracts to perform the translation reaction, those derived from E.coli, wheat germ, and rabbit reticulocyte are commercially available.For an E. coli extract, S-30 cell-free extract reported by Zubay et al.(e.g., see Non-Patent Reference 1) is commonly used. For preparing theE. coli S-30 extract, RNase I-defective strains such as A19 and D10 areused. However, when the target protein is sensitive to proteolyticdegradation, E. coli strain B, which is defective in ompT endoproteaseand ion protease activities may be used.

For synthesizing mRNA from a cloned cDNA, the cDNA must be introducedinto a suitable vector having various promoters. For increasing theprotein expression efficiency, intensive promoters for phage-derivedpolymerases such as T7, T3, and SP6 are used at present, and varioussystems suitable to various types of template DNAs are commerciallyavailable. Using such cell-free systems enables cloned DNA expression inan extremely simplified manner and enables cytotoxic protein synthesis.

However, it is known that systematic and comprehensive expression of alarge number of genes obtained from the recent genome analysis resultsin the existence of genes of relatively low expression and genes of noexpression. It is believed that the low expression of genes may becaused by the reduced efficiency in the translation stage based on thedifference in the nucleotide sequence as compared with the genes ofhigher expression.

On the other hand, it is known that antibiotics-resistant actinomycetesstrains have a property of enhanced producibility of secondarymetabolites (antibiotics, etc.), and it is reported that these arederived from the point mutation of ribosomal protein genes. It issuggested that, in these mutants, the conformation of the 16S ribosomalRNA (hereinafter referred to as “16SrRNA”) might change due to the pointmutations of the ribosomal protein S12 and S4, which influence the mRNAreading efficiency (e.g., see Non-Patent Reference 2), but themechanism, by which the mutation of the ribosomal protein enhances theproduction yield of the secondary metabolites of actinomycetes, is notyet clarified. It is reported that actinomycetes resistant tostreptomycin, gentamycin and rifampicin have a property to express anextremely increased amount of a specific transcriptional regulatoryprotein in the growth stage (e.g., see Non-Patent Reference 3, FIG. 5).However, nothing has been clarified as yet, relating to the exogenousprotein producibility in the extract of such actinomycetes cells. Ascompared with other bacteria, the growth speed of actinomycetes is slowand the optimum cultivation temperature thereof is low, and thus, it isdifficult to provide a large quantity of cell extract of actinomycetes.

[Non-Patent Reference 1]

-   Geoffrey Zubay, Annual Review of Genetics, 1973, Vol. 7, pp.    267-287.

[Non-Patent Reference 2]

-   Yoshiko Hosoya & 3 others, Antimicrobial Agents and Chemotherapy,    1988, Vol. 42, pp. 2041-2047.

[Non-Patent Reference 3]

-   Haifeng Hu & 1 other, Applied and Environmental Microbiology, 2001,    Vol. 67, pp. 1885-1892.

DISCLOSURE OF THE INVENTION

Therefore, it is an object of the present invention to increase theprotein producibility by improving the codon reading efficiency of mRNAon ribosome in a cell-free protein synthesis system using an E. colicell extract.

We, the present inventors, have studied the culture characteristics ofstreptomycin-resistant E. coli strains and the protein synthesisactivity of the extract thereof, and, as a result, have found that thecell extract prepared from E. coli having a mutation in a specific siteof the ribosomal protein S12 has an extremely high protein synthesisactivity. On the basis of this finding, we have completed the presentinvention.

According to a first aspect of the present invention, there is provideda cell extract prepared from E. coli having a mutation in the ribosomalprotein S12 gene.

In a preferred embodiment of this aspect of the invention, the mutationconfers a resistance or dependence to streptomycin on E. coli. Morepreferably, the mutation causes an amino acid substitution in theribosomal protein S12, and the amino acid substitution is at position 43of the amino acid sequence represented in SEQ ID NO:2. Even morepreferably, the amino acid substitution is a substitution from lysine tothreonine. Owing to the ribosomal protein S12 having the amino acidsubstitution, the mutant strain can grow even in the presence ofstreptomycin and its cell extract exhibits high protein synthesisactivity.

In another aspect of the present invention, there is provided a kit forcell-free protein synthesis, which comprises an extract for cell-freeprotein synthesis prepared from E. coli having a mutation in theribosomal protein S12 gene, and a mixture comprising an energyregenerating system, at least one amino acid, nucleotide triphosphate,and/or an RNA polymerase. The mixture may be mixed with the E. coliextract just before the protein synthesis reaction, but may bepreviously mixed with it. Accordingly, in still another aspect thereof,the invention provides a mixture for cell-free protein synthesis, whichcomprises an extract for cell-free protein synthesis prepared from theabove-mentioned E. coli, and at least one selected from the groupconsisting of an energy regenerating system, at least one amino acid,nucleotide triphosphate, and an RNA polymerase.

In still another aspect thereof, the invention further provides aprocess for producing a protein in a cell-free system. The processcomprises preparing a polynucleotide that encodes a protein, andexpressing the polynucleotide by the use of the extract for cell-freeprotein synthesis that is prepared from E. coli having a mutation in theribosomal protein S12 gene. In some case for expressing a desiredpolynucleotide, the expression efficiency may be sometimes extremely lowdepending on the nucleotide sequence of the target polynucleotide, forexample, due to any problems of the codon-anticodon pairing on theribosome. The process of the invention solves the problem by introducinga specific mutation into the ribosomal protein S12, and results in animprovement of the expression efficiency of the polynucleotide that isresistant to express.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows growth curves of wild-type E. coli and E. coli having amutation in the ribosomal protein S12, in terms of the culture turbidity(OD₆₀₀) relative to the culture time. All strains were cultivated in2×YT medium at 37° C.

FIG. 2 shows the results of CAT synthesis by the use of an extract ofwild-type E. coli and that of E. coli having a mutation in the ribosomalprotein S12. CAT synthesis was carried out according to a batch processat 37° C. for 1 hour.

FIG. 3 shows the results of analysis through sucrose density gradientultracentrifugation of S-30 extract of wild-type E. coli and E. colihaving a mutation in the ribosomal protein S12 in the presence of Mg²⁺of a different concentration.

FIG. 4 shows growth curves of wild-type E. coli and E. coli havingK88E-mutation in the ribosomal protein S12, in terms of the cultureturbidity (OD₆₀₀) relative to the culture time.

FIG. 5 shows the results of CAT synthesis by the use of S-30 extractprepared from the cells of wild-type E. coli and that from the cells ofE. coli having a mutation in the ribosomal protein S12 in differentgrowth stages (L, S1 and S2)

BEST MODE FOR CARRYING OUT THE INVENTION

Preferred embodiments of the invention are described hereinunder withreference to the drawings.

(Ribosomal Proteins 512 Mutants, and their Antibiotic ResistanceMechanism)

The extract of E. coli cells of the invention is prepared by cultivatinga mutant E. coli strains having a mutation in the ribosomal protein S12and extracting the mutant cells. The ribosomal protein 812 is a type ofribosome-constitutive protein. It is known that, in E. coli, theribosomal protein S12 binds with 16SrRNA to form a small subunit. Forexample, the amino acid sequence of protein S12 that constitutes theribosome 308 of E. coli is registered in Acc. No. P02367 in SWISS-PROT,and the amino acid sequence is shown in SEQ ID NO:2.

A large complex comprising protein and RNA, that is, ribosome catalyzesvarious processes in the stage of protein translation. The structure andthe function of the ribosome are very similar in both prokaryotes andeukaryotes, and pairs of small subunits gather to form a complex ofmillions of Daltons. The small subunit controls the binding of mRNA andtRNA, and the large subunit catalyzes the formation of peptide bond.

The elongation of polypeptide chain on ribosome includes binding of anaminoacyl tRNA to the site A on ribosome thereby causing base pairingwith three nucleotides of the mRNA having appeared in that site. Next,the carboxyl terminal of the polypeptide chain binding to the tRNAmolecule at the site P adjacent to the site A is left away, and apeptide bond is thereby formed by the action of the amino acid bindingto the tRNA molecule at the site A and the peptidyl transferase.Finally, the ribosome moves by three nucleotides along the mRNA, and ittransfers the peptidyl tRNA newly formed at the site A to the site P.

It is known that aminoglycoside-type antibiotics such as streptomycinand hygromycin specifically reduce the translational proof readingactivity of ribosome and induce misreading of mRNA codons. In oneembodiment of the invention, the mutation of the ribosomal protein S12can be obtained by screening the mutants that can grow in the presenceof an antibiotic such as streptomycin. As a result, by altering itsstructure and function in protein translation, the mutant ribosomeacquires antibiotic resistance in one aspect, and significant influenceson the protein translation efficiency and proof reading activity inanother aspect.

In one embodiment of the invention, there is provided a mutant strainthat has a mutation of improving the protein translation efficiency inthe ribosomal protein S12. The mutation may be of any type that givesresistance to or dependence on streptomycin or any other type that doesnot give such resistance or dependence, so far as it improves theprotein synthesis activity of cell extract. Preferably, it has asubstitution at any of lysine 43, arginine 86, valine 87, lysine 88,aspartic acid 89, leucine 90, praline 91, glycine 92 or arginine 94 inthe amino acid sequence of SEQ ID NO:2, as attaining a favorable result.For example, lysine 43 may be substituted with any of other 19 types ofnatural amino acids, but is preferably substituted with a hydrophilicamino acid, more preferably with threonine for attaining high proteinsynthesis activity. Lysine 88 may also be substituted with any of other19 types of natural amino acids, but is preferably with a hydrophilicamino acid, more preferably with arginine for providing an advantageouseffect. Leucine 90 or arginine 94 substitution does not bring aboutresistance to streptomycin but may improve protein synthesis activity.The mutation of the ribosomal protein S12 screened in the presence of anantibiotic is generally for single amino acid substitution, butscreening with plural antibiotics enables a mutation with two or moreamino acid substitutions. Site-specific mutation and homologousrecombination to chromosomal. DNA, as is described hereunder, bringsabout the formation of a mutant E. coli having a desired amino acidsubstitution in the ribosomal protein 512.

In another embodiment of the invention, there is provided a mutant ofribosomal protein 512 that has an excellent protein synthesis activityin the stationary phase of E. coli. For preparing an E. coli 5-30extract, generally employed are cells in a logarithmic growth phase (logphase, exponential phase) having a high protein synthesis activity. Thelogarithmic growth phase means a phase where the number of bacteria orcells increases logarithmically. When cells are inoculated in a newvessel, they first take a period of time before they could adapt to thenew condition (lag phase), and then they grow more and more in thesubsequent logarithmic growth phase and the growth of the cells willsoon stop depending on the size of the vessel and on the degree ofdegradation of the culture medium, and thus the cells are in astationary phase. In general, the cells in the logarithmic growth phasehave a higher protein synthesis activity than those in the stationaryphase, but they require a larger amount of the culture medium than thatin the stationary phase for preparing a predetermined number of cellssince the cell concentration is low. Accordingly, if the S-30 extracthaving a high protein synthesis activity could be prepared from E. colicells in the stationary stage, then it could be an extremely efficientprocess. One example of the ribosomal protein S12 mutant having a highprotein synthesis activity in the stationary phase is a mutant (K88E)having the amino acid sequence of SEQ ID NO:2 where lysine 88 issubstituted with glutamic acid. The reason why the S-30 extract from thecells of the stationary-phase K88E mutant could have a higher proteinsynthesis activity than the wild strain may be presumed as follows: As aresult of investigation of the characteristics of the ribosome in theextracts, the ribosome in the stationary-phase cells of the K88E mutantis more stable than those of the parent strain and the protein synthesisactivity per unit ribosome of the mutant is higher than that of theparent strain.

Of the E. coli mutants, those capable of growing in the presence ofstreptomycin may have the possibility that the translational proofreading activity of the ribosome is improved, or that is, the mRNA codonrecognition thereof is more accurate. In general, the codon recognitionaccuracy means the reduction in the affinity for tRNA of the ribosome,and it is thereby believed that a correct tRNA could be more strictlyselected from similar tRNAs competitively bonding to the site A of theribosome. Accordingly, it is believed that the cell extract of the E.coli mutant of the invention is advantageous in that its proteinsynthesis amount increases due to the improvement in the translationefficiency thereof and its codon recognition accuracy also increases.

(Screening of E. coli Strain Having Mutation at the Ribosomal ProteinS12 Gene)

E. coli strains having a mutation at the gene encoding ribosomal proteinS12 (rpsL) may be screened in various methods. One method comprisesselecting an E. coli mutant capable of growing in the presence ofhigh-concentration streptomycin. The E. coli mutant of the type may beobtained, for example, by plating a suspension of E. coli cells, eitherdirectly as it is or after treatment with UV irradiation, to an agarmedium that contains from 5 to 100 times the minimal growth inhibitoryconcentration of streptomycin (50 to 1000 μg/ml) and harvesting thecolonies formed within 2 to 7 days. The E. coli strains applicable tothe process include E, coli. A19 (rna,met), BL21, BL21star, BL21codonplus.

Another method is as follows: An rpsL gene that has a desired amino acidsubstitution by introducing a site-specific mutation thereinto isconstructed, and this is cloned into a plasmid DNA and is introducedinto the chromosomal DNA of E. coli through homologous recombination.The rpsL gene of E. coli is already known, and its nucleotide sequenceis registered, for example, in the database of GenBank or DDBJ as AccNo. V00355. The nucleotide sequence that corresponds to the codingregion is shown in SEQ ID NO:1. A site-specific mutation is introducedinto the rpsL gene cloned from the chromosomal DNA of E. coli throughPCR, whereby an amino acid substitution mutation may be introduced intothe desired site of the ribosomal protein 512. The method of in-vitrointroduction of a site-specific mutation into a cloned DNA includes, forexample, a Kunkel method (Kunkel, T. A. et al., Methods Enzymol. 154,367-382 (1987)); a double primer method (Zoller, M. J. and Smith, M.,Methods Enzymol. 154, 329-350 (1987)); a cassette mutation method(Wells, et al., Gene 34, 315-323 (1985)); a mega-primer method (Sarkar,G. and Sommer, S. S., Biotechniques 8, 404-407 (1990)). Next, themutation-introduced DNA fragment is cloned into a plasmid vector forhomologous recombination. The vector has, for example, achloramphenicol-resistant gene as a selective marker. When the plasmidin which the rpsL gene with the site-specific mutation was inserted, isintroduced into E. coli having a DNA recombination capability, forexample, E. coli BL21, then it results in homologous recombination. Thedouble recombinant with recombination at two sites of the introducedplasmid and the chromosomal DNA is given streptomycin resistance and issusceptible to chloramphenicol. The two-stage screening provides an E.coli mutant that has the desired rpsL gene inserted into the chromosomalDNA (e.g., see Hosoya et al. (vide supra)).

(Preparation of an Extract from the Mutant E. coli Cells)

As an E. coli extract, the S-30 extract prepared by the similar methodof Zubay, et al. (supra) can be used. The E. coli. S-30 extract containsall the enzymes and factors from E. coli necessary for transcription andtranslation. Additionally, supplemental mixture can be added. A concreteS-30 extract is prepared by first culturing the E. coli and harvestingthe cells by centrifugation and the like. The recovered cells are washedand resuspended in the buffer, and then lysed or broken with a Frenchpress, glass beads, Waring blender, or the like. The insoluble matter ofdisrupted E. coli cells is removed by centrifugation and the supernatantis then combined with a pre-incubation mixture and incubated. While thisoperation degrades the endogenous DNA and RNA, it may further include astep of adding a calcium salt or microccocal nuclease to degradecontaminating nucleic acids. The extract is then dialyzed to removeendogenous amino acids, nucleic acids nucleosides etc., stored in liquidnitrogen or −80° C. after dispensing appropriate aliquots.

For performing a protein synthesis reaction, the 8-30 extract issupplemented with all or a portion of the followings: Tris-acetate,dithiothreitol (DTT), the NTPs (ATP, CTP, GTP, and UTP), phosphoenolpyruvate, pyruvate kinase, at least one amino acid (20 naturallyoccurring amino acids including derivatives thereof. In case of labelingthe protein with a radioisotope, rests of a radio-labeled amino acid areadded.), polyethylene glycol (PEG), folic acid, cAMP, tRNA, ammoniumacetate, potassium acetate, potassium glutamate, an optimizedconcentration of magnesium acetate, and the like. These supplementalsolutions are usually stored separately from the S-30 extract, and thencombined just before use. Alternatively, the reaction mixture can bemade by combining an S-30 extract and supplemental mix, freezing andthawing the combination to remove the RNA degradsomes (see InternationalPublication WO 01/83805).

In the present invention, an energy regeneration system may preferablybe a combination of 0.02 to 5 μg/μl creatine kinase (CK) and 10 to 100mM creatine phosphate (CP), to which it is not limited, and any knownsubstance may be employed, such as a combination of 1 to 20 mMphosphoenol pyruvate (PEP) and 0.01 to 1 μg/μl pyruvate kinase and thelike. Any of PK and CK is an enzyme which regenerates an ATP from anADP, and requires PEP and CP as respective substrates.

The cell extract or supplemental mix can be distributed as a producteasy for use in aliquots. These products can be stored at frozen ordried state, and distributed as a kit in suitable containers for storageand for shipment to users. The kit can be accompanied by an instructionmanual, a positive control DNA, a vector DNA and the like.

(Protein Synthesis in Cell-Free Protein Synthesis System)

Protein synthesis in a cell-free protein synthesis system includes asystem where transcription and translation are separately effected indifferent test tubes like that for the nucleus and the cytoplasm ofeukaryotic cells, and a transcription/translation coupling system wherethe two are effected simultaneously. In the method of protein productionof the invention, an extract of prokaryotic cells, E. coli is used, andthe method is applicable to any of such reaction systems. For theinvention, however, the transcription/translation coupling system ispreferred in which any unstable mRNA is not directly processed.

First, a polynucleotide that encodes the protein to be expressed isprepared. In the invention, the “protein” may be any of a full-lengthprotein having a biological activity or a polypeptide fragment of a partof such a full-length protein. Many proteins, especially many eukaryoticproteins have plural domain structures as a result of evolution throughgene duplication, in which each domain has a structure and a functioncharacteristic of itself. Accordingly, it is extremely effective toexpress such proteins according to the method of the invention. Thenucleic acid that encodes the intended protein may be either DNA or RNA,and it may be extracted from the cells or tissues of eukaryotes orprokaryotes in any known method. DNA cloned from a cDNA library and thelike according to a known method is also employable herein.

For in-vitro synthesis of mRNA from a cloned cDNA, for example,employable is a phage-derived transcriptional system such as that fromT7, T3 or SP6, or an E. coli-derived transcriptional system. For mRNAsynthesis by the use of the system, employable are anycommercially-available kits such as MEGAscript (Ambion's trade name) andRiboMAX (Promega's trade name). The distance and the base sequence ofthe 5′-non-translational region between the translation initiation codon(ATG) and the promoter are important, and the region must include the SDsequence of E. coli. After a desired gene has been inserted into asuitable vector, the plasmid DNA is purified according to an alkali-SDSmethod or by the use of a DNA-coupling resin. Alternatively, using aprimer that contains the promoter, the DNA fragment amplified throughPCR may be used as a template. This is an extremely simple and rapidmethod for simultaneously processing different types of samples. In atranscription/translation coupling system, the plasmid DNA preparedherein or the PCR product can be directly used as the template in thecell-free protein synthesis system.

A semi-batch process where a small-molecule substrate such as ATP, GTP,amino acid or creatine phosphate is supplied to the reaction mixture viaa dialytic membrane or an ultrafilter while the waste is removed; and acontinuous process where the product is also removed via the membraneare reported. According to these processes, the reaction time may beprolonged by 5 to tens times, and a large quantity of protein can besynthesized. For example, according to a Kigawa et al's process (Kigawa,T., et al., FEBS Letters 1999, 442:15-19) or to the method described inJP-A 2000-175695, the above-mentioned reaction mixture is introducedinside the space of a dialytic membrane that has a molecular cutofflevel of at least 10,000, preferably at least 50,000, and this isdialyzed against a dialytic external liquid (including amino acid,energy source) of from 5 to 10 volume times the reaction mixture. Thedialysis is carried out generally at 20 to 40° C., preferably at 23 to30° C. with stirring, and at the time when the reaction speed islowered, the dialytic external liquid is exchanged with a fresh one. Theproducibility in the optimized cell-free protein synthesis system is farhigher than that in an intracellular system as in E. coli cells.

EXAMPLES

The invention is described more concretely in the following Examples. Inthe Examples, an E. coli strain BL21 was used as a parent strain, andthe colonies having grown in the presence of streptomycin were screenedto give 9 mutants. These mutants were analyzed for their properties suchas protein synthesis activity.

Example 1 Mutation from E. coli BL21

Mutants from a parent strain, E, coil BL21 were screened for thosecapable of growing in the presence of streptomycin. 150streptomycin-resistant spontaneous mutants of E. coli. BL21 wereobtained, and these were sequenced for the full-length rpsL gene. As aresult, it was confirmed that about 80% of the resistant mutants have amutation in the rpsL gene. Nine such rpsL mutants were obtained, andtheir typical strains are shown in Table 1. In Table 1, SmR in thephenotype column means streptomycin resistance; and SmD meansstreptomycin dependence. The mutation site indicates the basesubstitution site in the nucleotide sequence shown in SEQ ID NO:1; andthe amino acid substitution indicates the amino acid substitution sitein the amino acid sequence shown in SEQ ID NO:2. The symbol for the baseand the amino acid is a one-letter abbreviation.

TABLE 1 List of Mutants from E. coli BL21 Amino Acid Strain (Mutant)Mutation Site Substitution Phenotype BL21 — — wild type KO-365 A128GK43R SmR KO-368 A129C K43N SmR KO-371 A128C K43T SmR KO-374 A128T K43ISmR KO-375 A263G K88R SmR KO-376 C272T P91L SmR KO-378 C272A P91Q SmRKO-430 A262G K88E SmR KO-431 G275A G92D SmD

Example 2 Cultivation in the Presence of Antibiotic

Growth curves of E. coli mutant strains (K43T, K43R and K88R) obtainedin Example 1 and the parent strain (wild-type BL21) cultivated in 2×YTmedium at 37° C. were shown in FIG. 1, respectively In FIG. 1, theturbidity (600 nm absorbance) of the cultures sampled within a period offrom 2 hours to 9 hours after the start of the cultivation was measured,and used to estimate the amount of cells. Any of the mutant strainsshown in FIG. 1, showed the almost same growth pattern as that ofwild-type, whereas the amount of cells of the mutant K43T was thelargest in 8 hours after the start of the cultivation.

Example 3 Preparation of Cell Extract from E. coli Mutants, andComparison of Protein Synthesis Activity by CAT Assay

The mutant strains shown in Table 1 were separately cultivated in 2×YTmedium (16 g/liter of bactotrypsin, 10 g/liter of yeast extract, and 5g/liter of NaCl) at 37° C., and the cells in the mid-log phase (OD₆₀₀=3,about 10⁹ cells/ml) were collected. According to the method of Zubay etal. (vide supra), an E. coli extract S-30 was prepared from these cells.Protein synthesis was carried out as follows: To a solution having thecomposition shown in the following Table 2, added was 120 ng of pK7-CAT(CAT expression vector; see Kim et al., Eur. J. Biochem. 239, 881-886,1996). 7.2 μl of the E. coli 2-30 extract was added to it to make 30 μlas a whole. The reaction solution was batchwise incubated at 37° C. for1 hour. Thus synthesized, the CAT protein was quantified according tothe method of Shaw et al. (see Methods Enzymol., 735-755, 1975). Theresults are shown in FIG. 2.

TABLE 2 Ingredients Concentration HEPES-KOH pH 7.5 58.0 mMDithiothreitol 2.3 mM ATP 1.2 mM CTP, GTP, UTP 0.9 mM each Creatinephosphate 81.0 mM Creatine kinase 250.0 μg/ml Polyethylene glycol 80004.00% 3′,5′-cAMP 0.64 mM L(−)-5-Formyl-5,6,7,8-tetrahydrofolic acid 35.0μg/ml E. coli total tRNA 170.0 μg/ml Potassium glutamate 200.0 mMAmmonium acetate 27.7 mM Magnesium acetate 10.7 mM Amino acids (20types) 1.0 mM each T7RNA polymerase 16.0 units/μl

As in FIG. 2, it is understood that the amino acid substitution atposition 43 of the ribosomal protein S12 has a significant influence onthe protein synthesis activity. In particular, the protein synthesisactivity of the K43T mutant was about 1.3 times that of the wild type(WT) BL21. In the Table below FIG. 2, shown are the data of the proteinconcentration of the extract from each E. coli strain and the 260 nmabsorbance thereof (this essentially indicates the nucleic acidconcentration of the extract). From the data, it is understood thatthere is no direct relationship between the variations in the proteinamount and the nucleic acid amount in each extract and the proteinsynthesis activity of the extract.

Example 4 Characteristic Evaluation of Various E. coli Mutant Strains

At a fixed Mg²⁺ concentration of 20 mM or 5 mM, an S-30 extract wasprepared from wild-type E. coli and E. coli mutants (K43T, K43R) in thesame manner as in Example 3. The extract was put on a buffer (20 mMHEPES, 20 mM or 5 mM MgCl₂, 100 mM NH₄Cl, 4.5 mM 2-mercaptoethanol)containing from 6 to 38% of sucrose gradient, and centrifuged at 17000rpm for 17 hours by the use of a Beckman SW28 rotor. Then, this wasdivided into fractions of 0.8 ml each. In FIG. 3, the vertical axisindicates an estimated value of the ribosome-existing fraction throughpresumption of the 260 nm absorbance of the sample. As in FIG. 3, it isunderstood that, in the wild-type E. coli, a part of the 70S ribosomedissociated to give a 30S subunit at the low Mg²⁺ concentration of 5 mM.This is remarkable in the K43R mutant, in which a large part of the 70Sribosome dissociated into subunits 50S and 30S at the low Mg²⁺concentration of 5 mM. As opposed to this, the K43T mutant showed littledissociation of the 70S ribosome even at the low Mg²⁺ concentration.These results suggest that the K43T mutation in the ribosomal proteinS12 may stabilize the total ribosomal structure.

Example 5 Protein Synthesis from Various Mouse cDNAs

Using various mouse cDNAs as templates, protein synthesis was carriedout in an S-30 extract from wild-type and K43T mutant E. coli, in whichthe protein synthesis activity was evaluated. Three clones obtained froma mouse cDNA library (DDBJ Accession Nos. AK003622, AK010399 andAK019487) were used as templates. According to the two-step polymerasechain reaction (2-step PCR) a DNA fragment was prepared by adding a T7promoter, an SD sequence and a T7 terminator to the cDNA. First, thefirst PCR was carried out using the primer pair of the nucleotidesequence shown in Table 3.

TABLE 3   NUcleotide   Nucleotide   Sequence Sequence in forwardin reverse Template direction  direction  Clone cDNA primer primer Code(Acc. No.) (5′→3′) (5′→3′) A AK003622 CCAGCGGCTC CCTGACGAGG CTCGGGAATG GCCCCGAGTC  GAACCTTCTC ATCAGTCCTA TCTACA AAATTCAC (SEQ ID  (SEQ ID NO: 3) NO: 4) B AK010399 CCAGCGGCTC CCTGACGAGG CTCGGGAATG  GCCCCGAATT TTCCCAGAAC CATTAAAGCA AGCAG AACTTGTGAA (SEQ ID  (SEQ ID  NO: 5) NO: 6) CAK019487 CCAGCGGCTC CCTGACGAGG CTCGGGAGAG  GCCCCGAATT  TACAAAGCGGTTCAATTTCC GAGA CATAATCCTT (SEQ ID  (SEQ ID  NO: 7) NO: 8)

Each primer concentration was 0.25 μm. After the addition of DNApolymerase (by Rosh), 10 cycles at 94° C. for 30 seconds, at 60° C. for30 seconds and at 72° C. for 2 minutes each; 20 cycles at 94° C. for 30seconds, at 60° C. for 30 seconds, at 72° C. for 2 minutes plus 5seconds in every cycle; and finally one cycle at 72° C. for 7 minuteswere carried out.

Next, using the first PCR product obtained in the above reaction, a5′-primer having a histidine tag sequence downstream the T7 promotersequence:

(T7P-DM5-KH6, SEQ ID NO: 9)5′-GCTCTTGTCATTGTGCTTCGCATGATTACGAATTCAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGAGACCACAACGGTTTCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACATATGAAAGGCAGCAGCCATCATCATCATCATCACGATTACGATATCCCAACGACCGAAAACCTGTATTTTCAGGGATCCAGCGGCTCCTCGGG-3′,the 3′-primer having a T7 terminator sequence:

(T7T-DM3-Term, SEQ ID NO: 10)5′-CGGGGCCCTCGTCAGGATAATAATTGATTGATGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGATAACCTCGAGCTGCAGGCATGCAAGCTTGGCGAAGCACAATGACAAGAGC-3′,and a universal primer:

5′-GCTCTTGTCATTGTGCTTCG-3′, (U2, SEQ ID NO: 11)second PCR was carried out. The concentration of the 5′-primer and the3′-primer in the second PCR solution was 0.05 μM each, and theconcentration of the universal primer therein was 1 μM. Theamplification condition for the second PCR was the same as that for thefirst PCR, Next, the resulting DNA fragments were cloned into pPCR2.1 bythe use of TOPO TA-cloning kit (by Invitrogen) to thereby constructexpression vectors (clone A: P011114-10, clone B: P020107-17, clone C:P020408-01).

In the same manner as in Example 3, each expression vector of the clonesA to C was added to 30 μl of the reaction solution shown in Table 2 inthe concentration as indicated in Table 4, and batchwise processed at37° C. for 1 hour for protein synthesis therein. After the reaction,this was roughly purified with Ni-NTA agarose beads, and then subjectedto SDS-PAGE, and the protein was stained with a fluorescent dye SYPROOrange protein gel stains (by Molecular Probes). The band correspondingto the molecular weight of the synthesized protein was detected with aluminoimage analyzer LAS-1000 (by Fuji Photo Film), and quantified. Thusdetermined, the amount of the three proteins produced in the extract ofthe mutant K43T was compared with that produced in the wile type, andthe data are given in Table 4. It is understood that the amount of theprotein expressed by any of the clones A, B and C in the extract fromthe mutant K43T was larger by 1.5 to 2 times or more, than thatexpressed in the extract from the wild-type E. coli.

TABLE 4 Comparison of Synthesis Activity with Various Mouse cDNAExpression Vector Concentration in Clone Synthesis Molecular Weight ofRatio of Protein Code Reaction Solution Synthesized Protein ProductionAmount A   1 ng/μl 20726 1.58 B 2.3 ng/μl 22291 2 C   1 ng/μl 13549 2.2

Example 6

Mutant and wild-type (BL21) cells of E. coli in the mid-log phase (Lphase), stationary phase 1 (S1 phase) and stationary phase 2 (S2 phase)obtained in Example 1 were used to prepare cell-free extracts, and thesewere analyzed in point of their protein synthesis activity. Using anordinary liquid medium, the cells were cultivated to prepare seedcultures. The seed culture was inoculated into 7 liters of 2×YT mediumin a fermentor, and incubated therein with full aeration with stirringat 400 rpm at 37° C. This was periodically sampled out for 24 hoursafter the start of the incubation, and the turbidity of each sample (as600 nm absorbance) was measured, from which the amount of the cells wasestimated. As a result, it is understood that the growing speed of theK88E mutant was significantly lower than that of the wild-type E. coli,as in FIG. 4. In FIG. 4, the growth curves of the K88E mutant cultivatedwith or without streptomycin added thereto are shown. In both cases, thegrowing speed of the mutant cells was significantly low, and the amountof the mutant cells having grown within 24 hours is about a half of thatthe wild-type cells. The other mutants gave nearly the same growth curveas that of the wild-type strain.

The mid-log phase (L phase) of the K43R, K43N and K43T mutants and thewild-type strain (WT) was at OD600=3; and that of the K88E mutant isafter 6 hours from the start of cultivation. The S1 phase of all thestrains is after 9 hours from the start of cultivation; and the S2 phasethereof is after 24 hours from the start of cultivation. The cells inthese phases were collected, and an E. coli. 5-30 extract was preparedfrom these in the same manner as in Example 3, and the CAT protein masswas determined. The 260 nm absorbance of the S-30 extract was measured,and the amount of crude ribosome was obtained, and the CAT synthesisamount per unit ribosome is shown in FIG. 5. From the results in FIG. 5,it is understood that the CAT synthesis activity of the S-30 extractprepared from the L-phase cells of the K43T mutant was the highest, andthe CAT synthesis activity of the S-30 extract prepared from the52-phase cells of the K88E mutant was the highest. In FIG. 5, thevertical axis indicates the value obtained by dividing the CAT synthesisamount (μg) by the 260 nm absorbance (A260) for the crude ribosomeamount in the S-30 extract.

INDUSTRIAL APPLICABILITY

The cell extract prepared from the E. coli mutant of the invention has aspecific mutation at the ribosomal protein S12, and as compared withthat from wild-type E. coli cells, it shows a significantly higherprotein synthesis activity. There is a possibility that the ribosomalprotein S12 mutant may have an influence on the codon reading efficiencyof mRNA in the translation stage, and therefore there is a probabilitythat the mutant may have the ability to noticeably increase theexpression efficiency of mRNA of which the production amount was smallin a conventional method. In particular, of the E. coli mutants of theinvention, those capable of growing in the presence of streptomycin mayreadily prevent their culture from being contaminated with any otherexternal cells, and an extract for cell-free protein synthesis can beefficiently prepared from them.

1. An extract for cell-free protein synthesis, wherein said extract isprepared from Eshericia coli having a mutation at position 43 of theamino acid sequence represented in SEQ ID NO: 2 in the ribosomal proteinS12 gene encoding a SEQ ID NO: 2 variant from lysine to threonine thatprovides increased yield of protein in a cell-free protein synthesiscompared to the yield obtained in said cell-free protein synthesis usingan extract prepared from wild-type Eschericia coli.
 2. The extract ofclaim 1, wherein said increase in yield of the protein is from 1.5 to 2times that obtained from a cell-free protein synthesis using such anextract prepared from wild-type Eschericia coli.
 3. A kit for cell-freeprotein synthesis comprising: an extract prepared from Eschericia colihaving a mutation at position 43 of the amino acid sequence representedin SEQ ID NO: 2 in the ribosomal protein S12 gene encoding a SEQ ID NO:2 variant from lysine to threonine whereby the yield of protein from acell-free protein synthesis is increased compared to the yield obtainedin said cell-free protein synthesis using an extract prepared fromwild-type Eschericia coli, and a mixture comprising an energyregenerating system, at least one amino acid, ribonucleotidetriphosphates, and/or an RNA polymerase.
 4. A mixture for cell-freeprotein synthesis comprising an extract from Eschericia coli having amutation at position 43 of the amino acid sequence represented in SEQ IDNO: 2 in the ribosomal protein S12 gene encoding a SEQ ID NO: 2 variantfrom lysine to threonine, whereby the yield of protein from a cell-freeprotein synthesis is increased compared to the yield obtained in saidcell-free protein synthesis using an extract prepared from wild-typeEschericia coli, and at least one selected from the group consisting ofan energy regenerating system, at least one amino acid, ribonucleotidetriphosphates, and an RNA polymerase.